- Email: [email protected]
Nitrogen transfer from Phaseolus bean to intercropped maize measured using 15N-enrichment and 15N-isotope dilution methods
Soil Eiol. Lochem. Vol. 23. No. 4. pp. 339-346. 1991 Pnntcd in (ireal Encain. All rightsrncrvcd
0038-0717/91 53.00 + 0.00
Copyright I: 1991 PcrgamonPma pk
NITROGEN TRANSFER FROM PHASEOLUS BEAN TO INTERCROPPED MAIZE MEASURED USING “N-ENRICHMENT AND “N-ISOTOPE DILUTION METHODS KENNETH
E. GILLER, JUDY ORMESHER and FRU MARTIN AWAH
Department of Biochemistry and Biological Sciences. Wye College. University of London, Wye, Ashford, Kent, TN25 SAH. U.K. (Accepted
25 October
1990)
Summary-Transfer of N from Phasedus bean to intercropped maize was studied in glasshouse experiments using “N-foliar feeding and “N-isotope dilution methods. Nodulated and non-no&dating Phuseolus genotypes were included in separate treatments to help distinguish between benefits due to transfer of fixed N and competition for N in the growth medium. When intercropped with bean foliarly fed with (“NH,),SO,, maize was enriched with “N, showing that N had been transferred. The amounts of N transferred were small. and always < 5% of the N in the Nr-fixing beans. There was a &crease in shoot-N in maize intercropped with N,-fixing bean compared to maize intercropped with the non-nodulating beans. Non-nodulating bean transferred comparable amounts of N to intercropped maize plants although their total N content was less than a quarter of that in the N,-fixing beans. For the isotope dilution experiments, “N-fertilizer was incorporated into a soil-based compost together with sucrose to stabilise the “N-enrichment of available N. When plants grew vigorously no transfer of N from bean to maize was detected by isotope dilution, and again shoot N of maize intercropped with N,-fixing beans was less than that of maixc with non-nodulating beans. In a further experiment, growth of maize and bean plants was rcduccd by scvcrc insect attack and up to IS% (between 9 and I5 mg N pot-‘) of the N in N,-lixing beans was estimated by isotope dilution to have been transferred. Small (S-10 mg N pot -‘) but significant incrcascs in total N yield were found in the maize intercropped with N2-lixing bean compared to maize intcrcroppd with non-nodulating bean. In this experiment treatments with or without vesicular-arhuscular mycorrhiza were established but showed no significant ditferences in N-transfer from uninoculated plants. As transfer of N from the beans to intercropped cereals showed such little benefit under conditions of scvcrc N limitation, our results indicate that many careful field experiments are required before we can conclude that N-transfer from fhuseolus to intercropped cereals is significant in agriculture.
1NTRODUClION Intercropping of common bean and maize is a traditional and widely practiced cropping system in much of Latin America and also in parts of Africa (Adams et al., 1985). The benefits of intercropping are numerous, including more efficient capture and use of resources (e.g. light), economic advantages and “insurance” against crop failure (Willey, 1979). Attention has also been focused on the possible benefits of N,-fixation by grain legumes to intercropped cereals, which may be due to the legume fixing N2 solely for its own use and therefore affording less competition for soil N with the cereal, or by the legume contributing N directly for use by the intercropped cereal. The distinction bctwccn these two mechanisms by which legumes may improve the N nutrition of intercropped cereals has been acknowledged to be important. as the transfer of fixed N from the legume to the cereal may be viewed as a “facilitative” benefit, which may have the potential for further manipulation to increase cereal yields (Vandermeer, 1989). Many studies of the N economy found no evidence for transfer of N between legumes and cereals in intercrops
(e.g. Ofori
CI al.,
1987: van Kessel and 339
Roskowski. 1988) whereas other studies deduced significant amounts of transfer using “N-isotope dilution techniques (Eaglesham et al.. 1981). Much of the evidence used to support the idea of a significant transfer of N has been inferred from research on mixed grass-legume swards which persist in the field for much longer periods than most intercrops, but even here the evidence for or against the transfer of significant amounts of N is contradictory (cf. Vallis ef al., 1967; Ledgard ef al.. 1985; Ta and Faris. 1987) and may depend on the species in the mixture studied. There are a few direct measurements of N-transfer such as that of van Kessel ef al. (1985) who clearly demonstrated that transfer of “N from roots of soyabean to maize plants takes place, by using a split root system with “N-labelled soil to enrich the N in the legume plants. Here we describe experiments to measure N transfer in maizc-bean intercrops using a method in which the N of the intercropped legume was labelled with “N by foliar feeding with lJNammonium sulphate (Ledgard et al.. 1985) and also using a “N-isotope dilution method which has been used frequently to measure N-transfer between pasture legumes and grasses (Vallis ef al.. 1967). Experiments were carried out in glasshouse conditions with the aim of developing methods for later use in the
field, and treatments were included in which nonnodulating bean genotypes (Davis et al., 1988) were used to assess the role of root nodule loss in N transfer and to take account of inter-specific competition for soil N. Treatments in which plants were inoculated with vesicular-arbuscular mycorrhiza (VAM) were included, as direct connections between roots by fungal hyphae have been implicated in interplant nutrient transfer (Francis Edal., 1986) and VAM have been shown to increase the transfer of ‘rN from soyabean to intercropped maize (van Kessel et ol., 1985). In the experiments to explore the use of isotope dilution for measurement of N transfer, the soil was labelled with immobilised “N-fertilizer as this has been shown to reduce problems associated with differing patterns of soil and fertilizer-N uptake by crops (Witty and Ritz, 1984; Giller and Witty, 1987) which have caused problems for estimation of N transfer in other experiments (Papastylianou, 1988). MATERIALS AND METHODS
E.vperimcntal
conditions
All of the experiments were conducted in a glasshouse with day-night temperatures of 25-18°C rcspcctivcly. The N,-fixing Phaseofus genotype was RI2 30 (from CIAT. Colombia) and the non-nodulating genotype of Phaseolus was RIZ 30 No. I25 which had earlier been obtained by chemical mutagcncsis (Davis ef ul.. 1988). The maize was a dwarf variety (Kitumani) from Tanzania, and all maize and bean pots were inoculated with a suspension of Rhi:ohiwn Ieguminosarum bv. phuseoli stmin CIAT 899 at sowing. Plants wcrc watcrcd with N-free nutrient solution (based on Hewitt, 1966) containing a rcduccd P concentration (20 mgP I-‘) once a week and othcrwisc with deionised water. All treatments were completely randomiscd, with five replicates. Mycorrhizal inoculum was prcparcd as washed spores and fine hyphac of GIomus moS$eue mixed with sterile sand and placed below the seed of maize and bean plants at sowing. Filtered inoculum mixed with sterile sand was placed below each of the VAM-free plants. E.~periments I and 2:Joliar applicurion of“N to assess transfer of nitrogen from legumes 10 cereuls
Two experiments that differed only in the growth medium used were made simultaneously. In Experiment I the plants were grown in perlite, an inerl medium that supplies virtually no mineral N. and in Experiment 2 plants were grown in montmorillonilc clay chips that can supply slightly more N than perlile. Two bean plants were intercropped with two maize plants in each pot with or without inoculation with mycorrhiza. A basal fertilizer addition of 100 mg N Pot -’ as KNO, was added to each pot at sowing to allow the maize plants to establish. An earlier expcriment had shown that this amount of N did not suppress nodulation of the intercropped beans. Tests were made to determine the best method of application of “N to the leaves to ensure incorporation into the plant. The most convenient and successful method of applying the solution was found to be as a 30 m M solution of ( “NH,):SO,, 99 atom% “N spread on to the surface of the unifoliolate lcaves
when the plants were 3 weeks old. The leaves were then covered with sealable polythene bags to avoid contamination and further solution was added daily for a week. This gave “N enrichments in the bean plants of cu. 0.450 atom% “N excess in shoots, 0.800 atom% “N excess in roots and 0.350 atom% excess in nodules after a further 5 weeks’ growth. Other methods tested that were found to be less useful were to immerse the leaves in small bottles of solution, but this required much more solution and gave similar final enrichments, or to inject solution into the petioles which resulted in little enrichment with “N and was subject to greater risk of contamination. Less concentrated solutions of (“NH,)rSO1 gave decreased enrichment of lsN in the plant parts. The “N solution was applied to one of the unifoliolate leaves of each of the bean plants 3 weeks after sowing as described above. Plants were harvested 8 weeks after sowing, and the maize and bean root systems were separated carefully and subsampled for assessment of mycorrhizal infection, and underground parts (comprising roots and nodules for the beans) were analysed separately. The leaves to which lJN had been applied were excluded from the samples. Experiments 3 and 4: measurements ‘sN-isotope dilution
of N transfer by
The growth medium was prepared by mixing togcthcr a soil-based compost with sand and montmorillonitc clay chips in equal quantities to improve the aeration and rcducc the total N content (0.13% N). A solution containing (“NH,),SO, (20.1 atom% “N) and sucrose calculated to have a C:N ratio of IO: I was added to give a total amount of “N of l5mg per 2 kg pot. The soil was then kept for 4 months before use. In Experiment 3 the trcatmcnts. both with or without mycorrhizal inoculation, were nodulating or non-nodulating bean gcnotypcs intercropped with maize, and in Experiment 4 an extra treatment was included in which maize was planted alone. Two bean plants and two maize plants, or four maize plants in the case of the sole crop, were sown in each pot containing 2 kg of the “N-labelled soil. The pots were watered to maintain the soil at 60% of its water holding capacity (whc) once per week with N-free nutrient solution, and otherwise with deionised water as required. After 8 weeks of growth the plants were harvested, and maize and bean roots separated and subsampled for assessment of infection with mycorrhiza. Shoots and underground parts were analysed separately. Analytical
merhods
All plant samples were dried at 60°C. weighed and ground for further analysis. The total N concentrations wcrc determined after a semi-micro Kjeldahl digestion using an automated indo-phenol blue method (Varley. 1966). Nitrogen in the digests was conccntratcd by a Conway micro-diffusion technique (Conway. 1939) and “N enrichments determined on a Micromass 622 mass spectrometer (VG Isogas. Cheshire, England). Mycorrhizal infection was assesscd by a gridline method after staining using a modification of the method of Phillips and Hayman (1970).
Nitrogen transfers in bean-maize intercrops
341
Calculations
For example
Nitrogen in maize derived from bean was calculated in two ways: first, N transfer was calculated following the equation given by Ledgard et al. (1985) where
% N in maize from fixation by bean
% N in bean transferred
+ bean 15Ncontent
x 100%
(1)
% N in maize derived from beans R maize = R bean roots
x 100%
Cukulr tions: is0 tape dilution experiments The proportion of N fixed by bean was calculated by comparison of the cnrichmcnts of the nodulated bean with that of the maize or nonnodulating bean. % N from N,-fixation R nodulated legume x 100% (3) ( R reference plant > (Rennie et al., 1978).
Similarly the proportion of N in the intercrop maize derived from NJ-fixation was calculated by comparison of the enrichments of maize from the various treatments.
(4)
% N in maize derived from bean R sole maize - R intercrop maize = ( R sole maize - R intercrop bean >
Shoot
Root
In our experiments the atom% lJN excess of maize intercropped with non-nodulating beans could also be substituted for R sole maize in equations (4) and (5). RESULTS
Foliar feeding experiments Mycorrhizal infection was barely detectable (< 5% of root length was infected) in Experiments 1 and 2. therefore treatments with and without mycorrhizal inoculation were combined for statistical analyses. This was probably due to the inoculum being placed too close to the seed so that the roots wcrc able to “cscapc” infection. Growth and N accumulation of nodulatcd Phuseolus plants wcrc much greater than those of non-nodulatcd plants, indicating that N limited growth in both cxperimcnts (Tables I and 2). More mineral N was available for plant growth in the montmorillonitc medium (Experiment 2; Table 2) than in pcrlitc (Experiment I; Table I) as N accumulation of non-nodulating bean and of maize in montmorillonitc (> 125 mg pot-‘) was almost double that of the same treatments in perlite (68 mgpot-‘). Although in both experiments maize weighed twice as much as non-nodulating bean, the N accumulations were almost identical (Tables I and 2). Shoot N content of maize grown with N,-fixing beans (29 mg pot-‘) was less than that of maize in perlite grown with non-nodulating bean (38 mg pot-‘. Table I) perhaps due to intercrop competition for light from the much larger N,-fixing plants.
N content (me pole’) Shoot
0
(5)
and “N rccovcry of (a) bean and (b) maize plants grown as an inwcrop in pcrlitc and fed with (“NH,),SO, through the unifoliolate kaves of the beans (Experiment I) Dry weigh1 (e pal-‘)
x ,ooo/
(Ta and Faris, 1987).
1. Growth
Trcocments
x 100%
the proportion of N in the maize derived from bean (allowing that some of the bean N transferred would have been derived from the soil) was calculated as
(2)
(where R = atom% 15N excess) The amounts of N transferred were then calculated by multiplying these percentages with the relevant amount of N assimilated by the maize and beans. The amounts of N transfcrrcd wcrc then cxprcsscd as a % of total bcnn N transfcrrcd or as a % of N taken up by maize.
Table
R sole maize
(Vallis et al.. 1967).
(where the “N content is calculated as the total N content x atom %“N excess/lOO). Second, by assuming that the 15Nenrichment of the bean roots measured at final harvest was representative of the lJN enrichment of any N transferred to the maize. This enrichment was then used to calculate the percentage of N in the maize derived from the bean roots (as the % N derived from fertilizer or % N dff is often calculated)
= I-
R intercrop maize
to maize
Maize 15Ncontent = Maize “N content
=I-
Alom% “N excess
“N recovery (mg pot _’ 1
Rool
Shoot
Root
Shoot
Roar
0.424 I.725 0.118
0.512 1.336 0.067 NS
I.20 OS9 0.103
0.34 0.40 0.042
0.033 0.018 0.009 NS
0. I76 0.254 0.025 NS
0.01 0.01 0.002 NS
0.04 0.07 0.007
Maize + bean Maize + non-nod bean SE
6.2 I.8 0.27
2.5 2.1 0.16
(a) In bt2ln.r 265 64 38 30 19.1 3.7
Maize + bean Maize + non-nod bean SE
4.9 6.2 0.4s NS
3.7 4.2 0.19 NS
fb) In moire 29 29 38 30 2.7 I.9 NS
KESSETHE.
342
GILLER er al.
Table 2. Growth and “N recovery of (a) bean and (b) maize plants grown as an intercrop in montmorillonitc clay and fed with ( “NH,),SO, through the unifoliolate kaves of the beans (Experiment 2) Dry weight (g pot-‘) Treatments
Shoot
Maize + bean Ma& + non-nod bean SE Maize + bean Maize + non-nod bean SE
6.6 4.1 0.32 Il.0 II.9 0.63 NS
Root
N content (mg pot-‘) Shoot
dilution
Shoot
Root
2.4 3.3 0.13
(a) In beans 311 64 74 53 12.0 4.6
0.580 I.640 0.200
0.514 0.082
1.76 I .20 0.153
0.33 0.52 0.048
4.9 5.0 0.30 NS
(b) In maix 84 88 5.0 NS
0.024 0.012 0.007 NS
0.118 0.191 0.034 NS
0.0: 0.01 0.005 NS
0.05 0.08 0.002 NS
experiments
RWl
41 38 2.4 NS
I.021
(Table 4). The NZ-fixing plants accumulated more than three times as much N as non-nodulating bean or maize plants. The much smaller ‘5N-enrichments of the N*-fixing bean demonstrated clearly that this additional N was from N,-fixation. The nonnodulating plants had a significantly smaller weighted mean enrichment (0.634 atom% lsN excess) than the maize which had similar enrichments whether intercropped with the N,-fixing (0.691 atom% “N excess) or non-nodulating bean (0.705 atom% “N excess). Thus transfer of fixed N from bean could not be detected from 15N-enrichments of maize, and the only significant effect of intercropping maize with the ditierent bean treatments was that shoot N was less whcrc maize was grown with N,-fixing bean. Whether N,-fixation was calculated using non-nodulating bean or maize as a refcrcncc, similar results wcrc obtained indicating ca. 60% of the N came from N,fixation (Table 5). The diKcrcncc in ‘SN-enrichments between non-nodulating bean and maize grown in the same pot indicated that 10% of the N in non-nodulating bean had come from N,-fixation (Table 5). In Experiment 4, inoculation with VAM in a layer 3cm below the seed rcsultcd in infection of roughly 20% of the root length of beans and 40% of the root length of maize (Table 6). A small amount of mycorrhizal infection was also found in some uninoculated sole maize plants. All of the plants grew poorly in this experiment due to attack by thrips (Frankliniellu occidentalis) which caused obvious leaf damage particularly in beans. N,-fixation in nodulated bean resulted in a much larger N content and smaller lJN-enrichment than in non-nodulating bean or maize (Table 6). Sole maize plants had larger shoot and root weights and N contents in total, but this represents the total from four plants of maize whereas
Table 3. N transferred from beans to intercropped maize calculated using two methods after folk (Experiments 1 and 2)
N transferred as % of maize N
10.3 5.8 0.98
3.2 9.4 0.90
19.9 9.2 2.52
13.4 8.5 3.0 I
3.7 7.3 1.37
Il.0 6.5 2.5
N transferred as % of bean N
N transferred as % of maize N
Amount of N transferred
2.1 5.2 0.51
I.2 10.5 2.0
4.2 7.6 0.64 3.7 5.0 I .03
I.3 5.6 0.79
to the bean plants
N trwsfcrrcd 8s % of bean N
Amount of N transferred (ma Dot-‘)
Expdmrnf 2 in montmorillonifr Maize + bean 4.5 Maize + non-nod bean 6.5 SE 1.26
fccding (“NH,)$O,
(2) N trsnsfcr culculrtcd as % N derived from bean roots (Equation (2)]
(I) N transfer calculated using the method of Ledgard rf cl/.. 1985 [Equation (I)]
Experimcnc 1 In pcrlhr Maize + bean Maize + non-nod bean SE
(me pot-‘)
Root
Establishment of the mycorrhizal inoculum was poor and resulted in virtually no root infection in Experiment 3, therefore treatments with and without inoculation were again pooled for statistical analyses. Shoot weights of the maize plants were much larger than those of the beans although N contents of the maize and non-nodulating beans were similar
Treatment
“N recovery
Shoot
‘JN-enrichment of non-nodulating bean was < 2 atom% 15N excess in all cases, but much larger than that of N,-fixing bean (co.600 atom% “N excess) where the dilution effect of fixation of atmospheric “N2 was apparent (Tables 1 and 2). The total amount of “N incorporated through the unifoliolate leaves was similar in non-nodulating (2.09 mg N pot-‘) and N,-fixing bean (I .72 mg N pot-‘) in montmorillonite. but in perlitc N,-fixing bean (I.54 mg N pot-‘) absorbed more (0.99 mg N pot-‘) than non-nodulating bean which grew very poorly due to N deficiency. Transfer of N from bean to maize was detectable from the 15N-enrichment of all shoots and roots of maize but the amounts of “N rccovercd in the maize were small (~0. I mg N pot -I). Calculation of amounts of N transfcrrcd using equations (I) and (2) above gave diffurcnt results (Table 3). The amounts were greater if calculated using equation (2) based on the measured enrichment of bean roots. Surprisingly, using equation (I) more N transfer was found from non-nodulated beans than from N,-fixing beans, but the rcvcrsc was found when equation (2) was used for the calculations. The calculated amounts of N transferred were never more than 4% of the N in the N,-fixing beans, but this amounted to 20% of maize N in perlite where the total N accumulated by the plants was small (Table I). Isotope
Atom% “N excew
(mn
Dot-‘)
Nitrogen transfers in bean-maize intercrops
343
Tabk 4. Growth and N-uptake of (a) bean and (b) maize plants grown as an intercrop on ‘sN-labelkd soil (Experiment 3) Dry weight tg pot - ’ 1 Treatment
N content
Atom% lrN
(mgpot-‘)
CXceU
Root
Shoot
Root
Wcightcd mean atom% excess
I.8 1.8 0.07
(a) In bealu 297 58 79 24 II.5 2.4
0.250 0.656 0.009
0.300 0.562 0.012
0.258 0.634 0.008
2.2 2.3 0.13 NS
(b) In make 71 15 80 I5 2.7 0.9 NS
0.715 0.728 0.019 NS
0.584 0.585 0.016 NS
0.69 I 0.705 0.015 NS
Shoot
Root
Maize + bean Maize + non-nod bean SE
6.4 4. I 0.18
Maize + bean Maize + non-nod bean SE
10.2 II.3 0.51 NS
Shoot
iments. as the roots and shoots of maize plants were enriched with rsN (Tables 1 and 2). Calculation of the amounts of N transferred by either of the methods used here involves assumptions. The method of Ledgard et al. (1985) assumes that all of the “N absorbed by the beans is recovered in the plant parts of the bean and intercropped maize. The second method described. in which transfer is calculated by a method similar to the calculation of N derived from fertilizer (% N dfi’). assumes that the ‘sN-enrichment of bean roots at the time of harvest is representative of the r5N-enrichment of all the N available for transfer from beans to maize throughout the growth of crops. Further. both methods assume that the “N incorporated into the bean plants when 3 weeks old has an equal opportunity to be transferred to the maize as all other N in the plants. None of these assumptions is likely to fully apply. In particular. if the “N incorporated through the lcaves is utilised in structural components of the root or in the shoot, this will bc less likely to be lost to intercropped plants. For instance. N, fixed at later stages of plant growth may be more likely to be lost from the roots, but that would not be detected by these methods. Calculation of N transfer using equation (2) probably tended to overestimate N transfer, particularly where the beans wcrc fixing N, and thus had a smaller lsN-enrichment in their roots, whereas equation (I) of Ledgard et al. (1985) may well underestimate transfer. The larger proportions of maize N from transfer found when plants were grown in perlite (Table 3) were more a reflection of the severe N deficiency of the maize plants than that more transfer was taking place, as more N was actually transferred to the plants grown
the other treatments contained only two maize plants per pot. If the N contents of the maize and non-nodulating beans from those intercrops are added the total N removed per pot (65 or 56 mg N pot-‘) was similar to the amount of N removed by the sole maize treatments (58 or 55 mg N pot-‘). “N-enrichments of non-nodulating beans were much smaller (~0.250 atom% “N excess) than those of maize plants grown in the same pots (>0.320 atom% “N excess) which would indicate that the non-nodulating beans had tixcd 23-28% of their N (Table 5) but as they formed no nodules this is not likely. Sole maize had similar rJN-cnrichmcnt to the maize intcrcroppcd with non-nodulating bean but cnrichmcnts of maize intcrcroppcd with Nz-fixing bean wcrc much smaller (Table 6) indicating that some of the fixed N had been transfcrrcd from bean to maize. Calculation of N transfer using the equations of Vallis PI al. (1967) and Ta and Faris (1987) indicated that 20 to 35% of the maize N had been dcrivcd from intercropped N,-fixing bean, which amounted to IO-15% of the bean N (Table 7). Enrichments of maize plants intcrcroppcd with N,-fixing bean were reduced, but not significantly less, where they had been inoculated with VA mycorrhiza. The atom% “N excessof maize intercropped with non-nodulating bean was very similar to that of the sole maize, indicating that little N had been transferred. DISCUSSION
Measurement of N-transfer by ‘jN-foliar feeding Transfer of N from bean to intercropped maize was easily detected in both of our foliar feeding exper-
Table 5. Nitrogen fixation in bexr plants grown intercropped with maize on “N-labelled soil calculated by isotope dilution using the maize or the non-nodulatina beans as the reference plant (Extw-iments 3 and 4) ?‘o N tired Treatments
Maize rcfcrcncc
Non-nod reference
Maize reference
58 0. I.3
64 IO I.2
211 0. 9.a
228 II 8.0
84 90 0. 0’ 2.8
89 92 28 23 4.8
a5 100 0. 0’ 9.7
90 103 7 5 9.6
Non-nod reference
Exprrimmt J Maize + bean Maize + non-nod bean SE Experiment 4 Maize + bean Maize + bean + VAM Maize + non-nod bean Maize + non-nod bean + VAM SE ‘Excluded from statistical analysis.
N fixed (mg pot -I’
KENNETHE. GILLERer of.
34.4
Table 6. Growth. mycorrhizal Infection and “N-uptake of (a) bean and (b) maize plants grown as sole or mtercrops oo “N-laklled with or without mycorrhizal inoculation (VAM) (Experiment 4)
N contmt (me pa-‘)
Dry WighI (8 PC-‘) Shoot
Root
Shoot
kan kan + VA41 non-nod bean non-nod bean + VAM
3.3 3.9 1.2 I.? 0.28
1.2 I.1 I .o I.0 0.10
74 86 I5 I3 7.1
Sole maile Sole maize + VAM Mauc + kan Maue + kan + VAM MUX + non-nod kan Maize + non-nod kan + VAM SE
6.4 5.7 4.0 3.5 4.1 2.7 0.34
4.7 5.0 3.2 31 3.3 2.4 0.37
30 27 20 ::
Maize Maize Maize Maize SE
+ + + +
soil
Atom?,'b ‘!S excess % VAM infection
Root
Shoot
Roar
Weighted mean atom% W excess
(a) In beanl 25 26 IO IO 2.8
O+ 18 o* 20 1.6
0.03 I 0.020 0.232 0.255 0.015
0.060 0.W 0.227 0.239 0.019
0.039 0.025 0.243 0.248 0.015
(a) In make* 28 28 24 24 20 15 18 1.4 2.2
5t 4Ot 0) 39 It 38 5.5
0.354 0.381 0.285 0.218 0.374 0.369 0.022
0.‘8 0.;8; O.‘Y 0.G 0.299 0.279 0.013
0.316 0.326 0.252 0.225 0.337 0.320 0.014
*Four maize plants per pot in sole maize treatments, two maize plants per pot in intercrop treatments. Walues excluded from statistical analysis.
in montmorillonite clay where N yields of the nonfixing plants were greater. Whatever the limitations of the methods, it is surprising that although both methods indicate that maize has derived N from bean. no benefit can be seen in increased N uptake by maize. Perhaps similar amounts of N are transferred from maize to bean, but this was not tested in our experiments. Thus N transfer has not contributed to bcttcr growth and N yield of maize despite the severe N limitation for growth imposed in thcsc expcrimcnts and the large amount of N fixed by the beans. Thcrc was no indication that the lcgumc nodulescontributed much directly to N transfer as amounts transfcrrcd wcrc similar with both N,-fixing and non-nodulated beans. and ths proportion of bean N transferred was much less in the case of NJ-fixing beans. i~~c~ll.~urc,r,lt,nt of N-rrctnsfer by ‘5NN-isoropedilution
NJ-fixing bean accumulated roughly three times the amount of N found in maize or non-nodulating bean, indicllting that the amount of N available from the soil was limiting (Tables 4 and 5). The small ‘!N-enrichments of the N,-fixing beans compared to the maize or non-nodulating beans clearly demonstrate that this extra N was from N2-fixation. Despite the large amount of N fixed in the bean plants no benefit in terms of increased N yield was found in the maize plants intercropped with them. In fact in one experiment the only significant difference detected between maize intercropped with the N,-fixing beans or the non-nodulating beans was that the maize had smaller shoot N when intercropped with N,-fixing
beans, presumably due to competition from the larger plants (Table 4). In this experiment the “Nenrichments of maize were virtually identical whether intercropped with N,-fixing or non-nodulating bean (difference ~2%) suggesting that there was no significant transfer of N. We were surprised to find that the “N-enrichment of the non-nodulating beans was much less than that of maize grown in the same pot in both isotope dilution experiments (Tables 4 and 6). The “N-fertilizcr had been added to the soil with the sucrose to stimulate immobilisation of the “N in the microbial biomass, well in advance of the experiment. This should have reduced any possible diferences in the “N-enrichment of N absorbed from the growth medium due to ditl’ercnccs in N uptake pattern of the two crops (Witty, 1983; Giller and Witty, 1987). The seed N content of the two crops was similar (nonnodulating beans 7.6 mg N seed-‘; maize 6. I mg N seed-‘) and cannot have caused the difference in enrichments. The lower “N-enrichment of the nonnodulating beans thus indicates that they were able to obtain N which was less highly enriched in “N, but the source of this N is unclear. We are confident that this was not from N?-fixation as nodules have never been found on roots of this non-nodulating genotype of beans in this or in numerous other glasshouse and field experiments. Perhaps the legume can stimulate mineralisation of N from less highly enriched organic N fractions in the soil, although it is surprising that the “N-enrichments differed so much considering that the roots were closely entangled throughout the
T.thle 7. Nitrogen transfcrrcd from kans to inlercropped maize plants with or withoul inoculation with VA mycorrhiza. calculated using eauations (3) and (4) (Experiment 4) N transferred calculated by using equation (3) (Vallis cf al.. 1967)
Maize Maize Maize Maize SE’
+ + + +
beans beans + VAM non-nod kans non-nod kans + VAM
Amount of N transferred (me pot-‘)
N transferred as % of kan N
N transferred as % of maize N
9 14 I -3 2.5
IO I4 3 -1 3.5
20 31 2
lSlandard error for the firs1 IWO treatments means only.
-1
4.7
N transferred calculated using equation (4) (Ta and Faris. 1987) Amount of N transferred (mg pot-‘) IO I5 -3 I 2.8
X transferred as % of kan N
N transferred as % of maize N
II I5 -13 I 3.5
22 33 -14 2 4.2
345
Nitrogen transfers in b*afi-maize intercrops soil. When non-nodulating bean or maize were used as reference plants for measurement of Nz-fixation in nodulated bean, as expected the estimates differed (Table 5). In this case as the proportion of N estimated to have come from N,-furation was high in both experiments, the difference of iJN-enrichment in the two reference crops had a relatively small effect on the estimates. Differences in the ‘JN-enrichment between reference plants would cause increasing disparity where smaller estimates of N,-fixation are found. Transfer of N from N,-fixing bean to maize can also be calculated by isotope dilution by comparison of the iJN-enrichments of the maize intercropped with N,-fixing bean with either the enrichments of the sole maize or maize intercropped with non-nodulating bean. In this experiment there was little difference in iJN-enrichment of the sole maize and maize intercropped with the non-nodulating bean, but based on the above observation we would favour the use of a cereal intercropped with a non-fixing legume in such experiments to take account of possible effects due to inter-specific competition for N. In this experiment where plant growth was poor due to insect attack, IO-IS% of the bean N, equivalent to 20-30% of the maize N. was estimated to have been transferred (Table 7). As c. 90% of the bean N was estimated to have come from N,-fixation in this experiment, there was little difference in the results obtained using the equations of Vallis cf al. (1967) which takes account of only fixed N transfcrrcd. and that of Ta and Faris (1987) which allows for all sources of the N transfcrrcd from beans (Table 7). Maize intcrcroppcd with N,-fixing bean accumulated slightly, but significantly. more N than maize with non-nodulating bean (Table 7) but no incrcasc in N-transfer due to the presence of VAM was detected by either isotope dilution or total N accumulation. The smaller net N accumulation than the benefit indicated from the isotope dilution calculations of N transfer could have been due to transfer of some N from maize to bean.
Mechanisms of N transfer
The foliar feeding experiments clearly demonstrate underground movement of “N from the beans to the maize within a few weeks. The term “excretion” has been used in the literature to describe such relatively rapid loss of N from the legume roots, but we would suggest, by analogy with carbon losses from roots, the term “rhizodeposition” may be more appropriate (Whipps and Lynch, 1985). It is not possible in such experiments to distinguish between N lost actively or passively in exudates, N losses due to death and senescence of sloughed off cells from roots and nodules and indeed direct transfer of N through mycorrhizal hyphae. In all of these cases the rates of loss and turnover of N in the rhizosphere are likely to be such that they could result in movement of N from the legume to the cereal in days rather than weeks. The use of the term “rhizodeposition” may help to clarify this by not attempting to distinguish active and passive losses of N. Significance of N-transfer in intercrops
Despite the severe N limitation imposed in all of these experiments which was apparent in the
restricted growth and N accumulatioo in all plants of maize and non-nodulating bean, no obvious benefits in total N accumulation of maize were found by intercropping with N,&xing bean. The sensitivity of the iJN-foliar feeding experiments was such that transfer of enriched N was readily detected, but the amounts of N transferred were small and in all cases ~5% of the Nr fixed by beans (Table 3). In the isotope-dilution experiments no transfer of N was detected except where growth of all the maize and bean plants was badly affected by insect infestation, and although up to 35% of the maize N was estimated to have come from N transfer there was no obvious benefit in N yield. Although our initial intention was to identify methods suitable for measurement of N transfer between bean and maize in the field, it seems likely that amounts transferred may be negligible unless a severe limitation is placed on the growth of the beans such as the insect attack seen here. In pot experiments with peas and barley, Wilson and Wyss (1937) only found significant increases in N yield of the cereal when the plant growth was reduced by shading. Many field studies on legumocereal intercrops have found no evidence for N transfer (e.g. Ofori ef al.. 1987; Rerkasem and Rerkasem, 1988) and our results show little benefit from N transfer under conditions of severe N limitation which should increase the demand of the cereal for legume N. Numerous, careful ticld studies will be required to determine how frcqucntly and under what conditions benefits occur due to transfer of N from legumes to intercropped cereals in agriculture. Ac&nowle&rmmr.r-Wc thank J. C. Fear for help with chemical analyses. The Overseas Dcvclopmcnt Administration provided part of the funding for this research.
REFERENCES
Adams M. W.. Coync D. P.. Davis J. H. C., Graham D. H. and Francis C. A. (1985) Common bean (Phuseolus uulguris L.) In Grain Lqume Crops (R. J. Summcrlleld and E. H. Roberts. Eds), pp. 433-476. Collins. London. Conway E. J. (1939) Microdrjiiion Ano1ysi.rand Volumerric Error. Crosby-Lockwood, London. Davis J. H. C.. Giller K. E.. Kipc-Nolt J. A. and Awah F. M. (1988) Non-nodulating mutants in common beans. Crop Science 28. 859-860. Eaglesham A. R. J.. Anyanaba A., Ranga Rao V. and Eskew D. L. (1981) Improving the nitrogen nutrition of maize by intercropping with cowpca. Soil Biology & Biochemislry 13, 169-171. Francis R.. Finlay R. D. and Read D. J. (1986) Vaicular-arbuscular mycorrhiza in natural vegetation systems IV. Transfer of nutrients in intra- and inter-specific combinations of host plants. New fhyrologisr 102, 103-111. Gillcr K. E. and Witty J. F. (1987) Immobilized “N-fertilizer sources improve the accuracy of field estimates of N,-fixation by isotope dilution. Soil Biology CcBiochemisrry 19, 459463. Hewitt E. J. (1966) Sand and Wurcr Culrure Merho& in rhe Study o/ Plunr Nurrbion. Commonwealth Agricultural Bureau, Famham Royal. van Kcsscl C. and Roskoski J. P. (1988) Row spacing effects on N,-fixation. N-yield and soil N uptake of intercropped cowpca and maize. Plum and Soil 111, 17-23.
KES~ZTH E. GIU+R er af.
346
van Kessel C., Singleton P. W. and Hoben H. J. (1985) Enhanced N-transfer from soybean to maize by vesicular arbuscular mycorrhizal (VAM) fungi. Pianr Physiology 79, 562-563.
Ledgard S. F.. Freney J. R. and Simpson J. R. (1985) Assessing nitrogen transfer from legumes to associated grasses. Soil Biology & Biochemisrry 17. 575-577.
Ofori F., Pate J. S. and Stem W. R. (1987) Evaluation of N,-fixation and nitrogen economy of a maize/cowpea intercrop system using “N dilution methods. Pfanr and Soil 102. 149-160.
Papastylianou I. (1988) The “N methodology in estimating N,-fixation by vetch and pea grown in pure stand or in mixtures with oat. Piunr and Soil 107, 183-188. Phillips J. H. and Hayman D. S. (1970) Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection, Transacrions ofrhe Brirish Myrologicul Sociery 55,
I 58-
I6
I.
Rennie R. J.. Rennie D. A. and Fried M. (1978) Concepts of “N usage in dinitrogen fixation studies. In lsoropes in Biological Dinirrogen Fixurion. pp. 107-I 30. International Atomic Energy Agency. Vienna. Rerkasem K. and Rerkasem B. (1988) Yields and nitrogen nutrition of intercropped maize and ricebean (V&IO unih&rn ffhumb.] Ohwi and Ohashi). Planr and Soil IOR. 151-162.
Ta T. C. and Faris M. A. (1987). Effects of alfalfa proportions and clipping frquencies on timothy-alfalfa mixtures. II. Nitrogen fixation and transfer. Agronomy Journal 79, 820-824. Vallis I., Haydock K. P.. Ross P. J. and Henzell E. F. (1967) Isotopic studies on the uptake of nitrogen by pasture plants. III. The uptake of small additions of “N-labelled fertilizer by Rhodes grass and Townsville luame. Ausrralian
JOWMI of Agriculrural
Research 18. 865-877.
Vandermar J. (1989) The Ecology of hercropping. Cambridge University Press, Cambridge. Varley J. A. (1966) Automated methods for the determination of nitrogen, phosphorus and potassium in plant materials. Analysr 91, 119-121. Whipps J. M. and Lynch J. M. (1985) Energy losses by the plant in rhizode position. ANI& Proceedings o/rhe Phyrochemical Sociery of Europe 26, 59-71. Willey R. W. (1979) Intercropping-its importance and research needs. Part I. Competition and yield advantages. Field Crop Absrracrs 32, I-IO. Wilson P. W. and Wyss 0. (1937) Mixed cropping and excretion of nitrogen by leguminous plants. Soil Science Sociery of America Proceedings 11, 289-297. Witty J. F. (1983) Estimating N,-fixation in the field using isN-labelled fertilizer: some problems and solutions. Soil Biology & Biochemisrry 15. 631-639. Witty J. F. and Ritz K. (1984) Slow-release “N fertilizer formulations to measure Nr-fixation by isotope dilution. Soil Bio1og.v & Biochemisrry 16. 657-661.
0038-0717/91 53.00 + 0.00
Copyright I: 1991 PcrgamonPma pk
NITROGEN TRANSFER FROM PHASEOLUS BEAN TO INTERCROPPED MAIZE MEASURED USING “N-ENRICHMENT AND “N-ISOTOPE DILUTION METHODS KENNETH
E. GILLER, JUDY ORMESHER and FRU MARTIN AWAH
Department of Biochemistry and Biological Sciences. Wye College. University of London, Wye, Ashford, Kent, TN25 SAH. U.K. (Accepted
25 October
1990)
Summary-Transfer of N from Phasedus bean to intercropped maize was studied in glasshouse experiments using “N-foliar feeding and “N-isotope dilution methods. Nodulated and non-no&dating Phuseolus genotypes were included in separate treatments to help distinguish between benefits due to transfer of fixed N and competition for N in the growth medium. When intercropped with bean foliarly fed with (“NH,),SO,, maize was enriched with “N, showing that N had been transferred. The amounts of N transferred were small. and always < 5% of the N in the Nr-fixing beans. There was a &crease in shoot-N in maize intercropped with N,-fixing bean compared to maize intercropped with the non-nodulating beans. Non-nodulating bean transferred comparable amounts of N to intercropped maize plants although their total N content was less than a quarter of that in the N,-fixing beans. For the isotope dilution experiments, “N-fertilizer was incorporated into a soil-based compost together with sucrose to stabilise the “N-enrichment of available N. When plants grew vigorously no transfer of N from bean to maize was detected by isotope dilution, and again shoot N of maize intercropped with N,-fixing beans was less than that of maixc with non-nodulating beans. In a further experiment, growth of maize and bean plants was rcduccd by scvcrc insect attack and up to IS% (between 9 and I5 mg N pot-‘) of the N in N,-lixing beans was estimated by isotope dilution to have been transferred. Small (S-10 mg N pot -‘) but significant incrcascs in total N yield were found in the maize intercropped with N2-lixing bean compared to maize intcrcroppd with non-nodulating bean. In this experiment treatments with or without vesicular-arhuscular mycorrhiza were established but showed no significant ditferences in N-transfer from uninoculated plants. As transfer of N from the beans to intercropped cereals showed such little benefit under conditions of scvcrc N limitation, our results indicate that many careful field experiments are required before we can conclude that N-transfer from fhuseolus to intercropped cereals is significant in agriculture.
1NTRODUClION Intercropping of common bean and maize is a traditional and widely practiced cropping system in much of Latin America and also in parts of Africa (Adams et al., 1985). The benefits of intercropping are numerous, including more efficient capture and use of resources (e.g. light), economic advantages and “insurance” against crop failure (Willey, 1979). Attention has also been focused on the possible benefits of N,-fixation by grain legumes to intercropped cereals, which may be due to the legume fixing N2 solely for its own use and therefore affording less competition for soil N with the cereal, or by the legume contributing N directly for use by the intercropped cereal. The distinction bctwccn these two mechanisms by which legumes may improve the N nutrition of intercropped cereals has been acknowledged to be important. as the transfer of fixed N from the legume to the cereal may be viewed as a “facilitative” benefit, which may have the potential for further manipulation to increase cereal yields (Vandermeer, 1989). Many studies of the N economy found no evidence for transfer of N between legumes and cereals in intercrops
(e.g. Ofori
CI al.,
1987: van Kessel and 339
Roskowski. 1988) whereas other studies deduced significant amounts of transfer using “N-isotope dilution techniques (Eaglesham et al.. 1981). Much of the evidence used to support the idea of a significant transfer of N has been inferred from research on mixed grass-legume swards which persist in the field for much longer periods than most intercrops, but even here the evidence for or against the transfer of significant amounts of N is contradictory (cf. Vallis ef al., 1967; Ledgard ef al.. 1985; Ta and Faris. 1987) and may depend on the species in the mixture studied. There are a few direct measurements of N-transfer such as that of van Kessel ef al. (1985) who clearly demonstrated that transfer of “N from roots of soyabean to maize plants takes place, by using a split root system with “N-labelled soil to enrich the N in the legume plants. Here we describe experiments to measure N transfer in maizc-bean intercrops using a method in which the N of the intercropped legume was labelled with “N by foliar feeding with lJNammonium sulphate (Ledgard et al.. 1985) and also using a “N-isotope dilution method which has been used frequently to measure N-transfer between pasture legumes and grasses (Vallis ef al.. 1967). Experiments were carried out in glasshouse conditions with the aim of developing methods for later use in the
field, and treatments were included in which nonnodulating bean genotypes (Davis et al., 1988) were used to assess the role of root nodule loss in N transfer and to take account of inter-specific competition for soil N. Treatments in which plants were inoculated with vesicular-arbuscular mycorrhiza (VAM) were included, as direct connections between roots by fungal hyphae have been implicated in interplant nutrient transfer (Francis Edal., 1986) and VAM have been shown to increase the transfer of ‘rN from soyabean to intercropped maize (van Kessel et ol., 1985). In the experiments to explore the use of isotope dilution for measurement of N transfer, the soil was labelled with immobilised “N-fertilizer as this has been shown to reduce problems associated with differing patterns of soil and fertilizer-N uptake by crops (Witty and Ritz, 1984; Giller and Witty, 1987) which have caused problems for estimation of N transfer in other experiments (Papastylianou, 1988). MATERIALS AND METHODS
E.vperimcntal
conditions
All of the experiments were conducted in a glasshouse with day-night temperatures of 25-18°C rcspcctivcly. The N,-fixing Phaseofus genotype was RI2 30 (from CIAT. Colombia) and the non-nodulating genotype of Phaseolus was RIZ 30 No. I25 which had earlier been obtained by chemical mutagcncsis (Davis ef ul.. 1988). The maize was a dwarf variety (Kitumani) from Tanzania, and all maize and bean pots were inoculated with a suspension of Rhi:ohiwn Ieguminosarum bv. phuseoli stmin CIAT 899 at sowing. Plants wcrc watcrcd with N-free nutrient solution (based on Hewitt, 1966) containing a rcduccd P concentration (20 mgP I-‘) once a week and othcrwisc with deionised water. All treatments were completely randomiscd, with five replicates. Mycorrhizal inoculum was prcparcd as washed spores and fine hyphac of GIomus moS$eue mixed with sterile sand and placed below the seed of maize and bean plants at sowing. Filtered inoculum mixed with sterile sand was placed below each of the VAM-free plants. E.~periments I and 2:Joliar applicurion of“N to assess transfer of nitrogen from legumes 10 cereuls
Two experiments that differed only in the growth medium used were made simultaneously. In Experiment I the plants were grown in perlite, an inerl medium that supplies virtually no mineral N. and in Experiment 2 plants were grown in montmorillonilc clay chips that can supply slightly more N than perlile. Two bean plants were intercropped with two maize plants in each pot with or without inoculation with mycorrhiza. A basal fertilizer addition of 100 mg N Pot -’ as KNO, was added to each pot at sowing to allow the maize plants to establish. An earlier expcriment had shown that this amount of N did not suppress nodulation of the intercropped beans. Tests were made to determine the best method of application of “N to the leaves to ensure incorporation into the plant. The most convenient and successful method of applying the solution was found to be as a 30 m M solution of ( “NH,):SO,, 99 atom% “N spread on to the surface of the unifoliolate lcaves
when the plants were 3 weeks old. The leaves were then covered with sealable polythene bags to avoid contamination and further solution was added daily for a week. This gave “N enrichments in the bean plants of cu. 0.450 atom% “N excess in shoots, 0.800 atom% “N excess in roots and 0.350 atom% excess in nodules after a further 5 weeks’ growth. Other methods tested that were found to be less useful were to immerse the leaves in small bottles of solution, but this required much more solution and gave similar final enrichments, or to inject solution into the petioles which resulted in little enrichment with “N and was subject to greater risk of contamination. Less concentrated solutions of (“NH,)rSO1 gave decreased enrichment of lsN in the plant parts. The “N solution was applied to one of the unifoliolate leaves of each of the bean plants 3 weeks after sowing as described above. Plants were harvested 8 weeks after sowing, and the maize and bean root systems were separated carefully and subsampled for assessment of mycorrhizal infection, and underground parts (comprising roots and nodules for the beans) were analysed separately. The leaves to which lJN had been applied were excluded from the samples. Experiments 3 and 4: measurements ‘sN-isotope dilution
of N transfer by
The growth medium was prepared by mixing togcthcr a soil-based compost with sand and montmorillonitc clay chips in equal quantities to improve the aeration and rcducc the total N content (0.13% N). A solution containing (“NH,),SO, (20.1 atom% “N) and sucrose calculated to have a C:N ratio of IO: I was added to give a total amount of “N of l5mg per 2 kg pot. The soil was then kept for 4 months before use. In Experiment 3 the trcatmcnts. both with or without mycorrhizal inoculation, were nodulating or non-nodulating bean gcnotypcs intercropped with maize, and in Experiment 4 an extra treatment was included in which maize was planted alone. Two bean plants and two maize plants, or four maize plants in the case of the sole crop, were sown in each pot containing 2 kg of the “N-labelled soil. The pots were watered to maintain the soil at 60% of its water holding capacity (whc) once per week with N-free nutrient solution, and otherwise with deionised water as required. After 8 weeks of growth the plants were harvested, and maize and bean roots separated and subsampled for assessment of infection with mycorrhiza. Shoots and underground parts were analysed separately. Analytical
merhods
All plant samples were dried at 60°C. weighed and ground for further analysis. The total N concentrations wcrc determined after a semi-micro Kjeldahl digestion using an automated indo-phenol blue method (Varley. 1966). Nitrogen in the digests was conccntratcd by a Conway micro-diffusion technique (Conway. 1939) and “N enrichments determined on a Micromass 622 mass spectrometer (VG Isogas. Cheshire, England). Mycorrhizal infection was assesscd by a gridline method after staining using a modification of the method of Phillips and Hayman (1970).
Nitrogen transfers in bean-maize intercrops
341
Calculations
For example
Nitrogen in maize derived from bean was calculated in two ways: first, N transfer was calculated following the equation given by Ledgard et al. (1985) where
% N in maize from fixation by bean
% N in bean transferred
+ bean 15Ncontent
x 100%
(1)
% N in maize derived from beans R maize = R bean roots
x 100%
Cukulr tions: is0 tape dilution experiments The proportion of N fixed by bean was calculated by comparison of the cnrichmcnts of the nodulated bean with that of the maize or nonnodulating bean. % N from N,-fixation R nodulated legume x 100% (3) ( R reference plant > (Rennie et al., 1978).
Similarly the proportion of N in the intercrop maize derived from NJ-fixation was calculated by comparison of the enrichments of maize from the various treatments.
(4)
% N in maize derived from bean R sole maize - R intercrop maize = ( R sole maize - R intercrop bean >
Shoot
Root
In our experiments the atom% lJN excess of maize intercropped with non-nodulating beans could also be substituted for R sole maize in equations (4) and (5). RESULTS
Foliar feeding experiments Mycorrhizal infection was barely detectable (< 5% of root length was infected) in Experiments 1 and 2. therefore treatments with and without mycorrhizal inoculation were combined for statistical analyses. This was probably due to the inoculum being placed too close to the seed so that the roots wcrc able to “cscapc” infection. Growth and N accumulation of nodulatcd Phuseolus plants wcrc much greater than those of non-nodulatcd plants, indicating that N limited growth in both cxperimcnts (Tables I and 2). More mineral N was available for plant growth in the montmorillonitc medium (Experiment 2; Table 2) than in pcrlitc (Experiment I; Table I) as N accumulation of non-nodulating bean and of maize in montmorillonitc (> 125 mg pot-‘) was almost double that of the same treatments in perlite (68 mgpot-‘). Although in both experiments maize weighed twice as much as non-nodulating bean, the N accumulations were almost identical (Tables I and 2). Shoot N content of maize grown with N,-fixing beans (29 mg pot-‘) was less than that of maize in perlite grown with non-nodulating bean (38 mg pot-‘. Table I) perhaps due to intercrop competition for light from the much larger N,-fixing plants.
N content (me pole’) Shoot
0
(5)
and “N rccovcry of (a) bean and (b) maize plants grown as an inwcrop in pcrlitc and fed with (“NH,),SO, through the unifoliolate kaves of the beans (Experiment I) Dry weigh1 (e pal-‘)
x ,ooo/
(Ta and Faris, 1987).
1. Growth
Trcocments
x 100%
the proportion of N in the maize derived from bean (allowing that some of the bean N transferred would have been derived from the soil) was calculated as
(2)
(where R = atom% 15N excess) The amounts of N transferred were then calculated by multiplying these percentages with the relevant amount of N assimilated by the maize and beans. The amounts of N transfcrrcd wcrc then cxprcsscd as a % of total bcnn N transfcrrcd or as a % of N taken up by maize.
Table
R sole maize
(Vallis et al.. 1967).
(where the “N content is calculated as the total N content x atom %“N excess/lOO). Second, by assuming that the 15Nenrichment of the bean roots measured at final harvest was representative of the lJN enrichment of any N transferred to the maize. This enrichment was then used to calculate the percentage of N in the maize derived from the bean roots (as the % N derived from fertilizer or % N dff is often calculated)
= I-
R intercrop maize
to maize
Maize 15Ncontent = Maize “N content
=I-
Alom% “N excess
“N recovery (mg pot _’ 1
Rool
Shoot
Root
Shoot
Roar
0.424 I.725 0.118
0.512 1.336 0.067 NS
I.20 OS9 0.103
0.34 0.40 0.042
0.033 0.018 0.009 NS
0. I76 0.254 0.025 NS
0.01 0.01 0.002 NS
0.04 0.07 0.007
Maize + bean Maize + non-nod bean SE
6.2 I.8 0.27
2.5 2.1 0.16
(a) In bt2ln.r 265 64 38 30 19.1 3.7
Maize + bean Maize + non-nod bean SE
4.9 6.2 0.4s NS
3.7 4.2 0.19 NS
fb) In moire 29 29 38 30 2.7 I.9 NS
KESSETHE.
342
GILLER er al.
Table 2. Growth and “N recovery of (a) bean and (b) maize plants grown as an intercrop in montmorillonitc clay and fed with ( “NH,),SO, through the unifoliolate kaves of the beans (Experiment 2) Dry weight (g pot-‘) Treatments
Shoot
Maize + bean Ma& + non-nod bean SE Maize + bean Maize + non-nod bean SE
6.6 4.1 0.32 Il.0 II.9 0.63 NS
Root
N content (mg pot-‘) Shoot
dilution
Shoot
Root
2.4 3.3 0.13
(a) In beans 311 64 74 53 12.0 4.6
0.580 I.640 0.200
0.514 0.082
1.76 I .20 0.153
0.33 0.52 0.048
4.9 5.0 0.30 NS
(b) In maix 84 88 5.0 NS
0.024 0.012 0.007 NS
0.118 0.191 0.034 NS
0.0: 0.01 0.005 NS
0.05 0.08 0.002 NS
experiments
RWl
41 38 2.4 NS
I.021
(Table 4). The NZ-fixing plants accumulated more than three times as much N as non-nodulating bean or maize plants. The much smaller ‘5N-enrichments of the N*-fixing bean demonstrated clearly that this additional N was from N,-fixation. The nonnodulating plants had a significantly smaller weighted mean enrichment (0.634 atom% lsN excess) than the maize which had similar enrichments whether intercropped with the N,-fixing (0.691 atom% “N excess) or non-nodulating bean (0.705 atom% “N excess). Thus transfer of fixed N from bean could not be detected from 15N-enrichments of maize, and the only significant effect of intercropping maize with the ditierent bean treatments was that shoot N was less whcrc maize was grown with N,-fixing bean. Whether N,-fixation was calculated using non-nodulating bean or maize as a refcrcncc, similar results wcrc obtained indicating ca. 60% of the N came from N,fixation (Table 5). The diKcrcncc in ‘SN-enrichments between non-nodulating bean and maize grown in the same pot indicated that 10% of the N in non-nodulating bean had come from N,-fixation (Table 5). In Experiment 4, inoculation with VAM in a layer 3cm below the seed rcsultcd in infection of roughly 20% of the root length of beans and 40% of the root length of maize (Table 6). A small amount of mycorrhizal infection was also found in some uninoculated sole maize plants. All of the plants grew poorly in this experiment due to attack by thrips (Frankliniellu occidentalis) which caused obvious leaf damage particularly in beans. N,-fixation in nodulated bean resulted in a much larger N content and smaller lJN-enrichment than in non-nodulating bean or maize (Table 6). Sole maize plants had larger shoot and root weights and N contents in total, but this represents the total from four plants of maize whereas
Table 3. N transferred from beans to intercropped maize calculated using two methods after folk (Experiments 1 and 2)
N transferred as % of maize N
10.3 5.8 0.98
3.2 9.4 0.90
19.9 9.2 2.52
13.4 8.5 3.0 I
3.7 7.3 1.37
Il.0 6.5 2.5
N transferred as % of bean N
N transferred as % of maize N
Amount of N transferred
2.1 5.2 0.51
I.2 10.5 2.0
4.2 7.6 0.64 3.7 5.0 I .03
I.3 5.6 0.79
to the bean plants
N trwsfcrrcd 8s % of bean N
Amount of N transferred (ma Dot-‘)
Expdmrnf 2 in montmorillonifr Maize + bean 4.5 Maize + non-nod bean 6.5 SE 1.26
fccding (“NH,)$O,
(2) N trsnsfcr culculrtcd as % N derived from bean roots (Equation (2)]
(I) N transfer calculated using the method of Ledgard rf cl/.. 1985 [Equation (I)]
Experimcnc 1 In pcrlhr Maize + bean Maize + non-nod bean SE
(me pot-‘)
Root
Establishment of the mycorrhizal inoculum was poor and resulted in virtually no root infection in Experiment 3, therefore treatments with and without inoculation were again pooled for statistical analyses. Shoot weights of the maize plants were much larger than those of the beans although N contents of the maize and non-nodulating beans were similar
Treatment
“N recovery
Shoot
‘JN-enrichment of non-nodulating bean was < 2 atom% 15N excess in all cases, but much larger than that of N,-fixing bean (co.600 atom% “N excess) where the dilution effect of fixation of atmospheric “N2 was apparent (Tables 1 and 2). The total amount of “N incorporated through the unifoliolate leaves was similar in non-nodulating (2.09 mg N pot-‘) and N,-fixing bean (I .72 mg N pot-‘) in montmorillonite. but in perlitc N,-fixing bean (I.54 mg N pot-‘) absorbed more (0.99 mg N pot-‘) than non-nodulating bean which grew very poorly due to N deficiency. Transfer of N from bean to maize was detectable from the 15N-enrichment of all shoots and roots of maize but the amounts of “N rccovercd in the maize were small (~0. I mg N pot -I). Calculation of amounts of N transfcrrcd using equations (I) and (2) above gave diffurcnt results (Table 3). The amounts were greater if calculated using equation (2) based on the measured enrichment of bean roots. Surprisingly, using equation (I) more N transfer was found from non-nodulated beans than from N,-fixing beans, but the rcvcrsc was found when equation (2) was used for the calculations. The calculated amounts of N transferred were never more than 4% of the N in the N,-fixing beans, but this amounted to 20% of maize N in perlite where the total N accumulated by the plants was small (Table I). Isotope
Atom% “N excew
(mn
Dot-‘)
Nitrogen transfers in bean-maize intercrops
343
Tabk 4. Growth and N-uptake of (a) bean and (b) maize plants grown as an intercrop on ‘sN-labelkd soil (Experiment 3) Dry weight tg pot - ’ 1 Treatment
N content
Atom% lrN
(mgpot-‘)
CXceU
Root
Shoot
Root
Wcightcd mean atom% excess
I.8 1.8 0.07
(a) In bealu 297 58 79 24 II.5 2.4
0.250 0.656 0.009
0.300 0.562 0.012
0.258 0.634 0.008
2.2 2.3 0.13 NS
(b) In make 71 15 80 I5 2.7 0.9 NS
0.715 0.728 0.019 NS
0.584 0.585 0.016 NS
0.69 I 0.705 0.015 NS
Shoot
Root
Maize + bean Maize + non-nod bean SE
6.4 4. I 0.18
Maize + bean Maize + non-nod bean SE
10.2 II.3 0.51 NS
Shoot
iments. as the roots and shoots of maize plants were enriched with rsN (Tables 1 and 2). Calculation of the amounts of N transferred by either of the methods used here involves assumptions. The method of Ledgard et al. (1985) assumes that all of the “N absorbed by the beans is recovered in the plant parts of the bean and intercropped maize. The second method described. in which transfer is calculated by a method similar to the calculation of N derived from fertilizer (% N dfi’). assumes that the ‘sN-enrichment of bean roots at the time of harvest is representative of the r5N-enrichment of all the N available for transfer from beans to maize throughout the growth of crops. Further. both methods assume that the “N incorporated into the bean plants when 3 weeks old has an equal opportunity to be transferred to the maize as all other N in the plants. None of these assumptions is likely to fully apply. In particular. if the “N incorporated through the lcaves is utilised in structural components of the root or in the shoot, this will bc less likely to be lost to intercropped plants. For instance. N, fixed at later stages of plant growth may be more likely to be lost from the roots, but that would not be detected by these methods. Calculation of N transfer using equation (2) probably tended to overestimate N transfer, particularly where the beans wcrc fixing N, and thus had a smaller lsN-enrichment in their roots, whereas equation (I) of Ledgard et al. (1985) may well underestimate transfer. The larger proportions of maize N from transfer found when plants were grown in perlite (Table 3) were more a reflection of the severe N deficiency of the maize plants than that more transfer was taking place, as more N was actually transferred to the plants grown
the other treatments contained only two maize plants per pot. If the N contents of the maize and non-nodulating beans from those intercrops are added the total N removed per pot (65 or 56 mg N pot-‘) was similar to the amount of N removed by the sole maize treatments (58 or 55 mg N pot-‘). “N-enrichments of non-nodulating beans were much smaller (~0.250 atom% “N excess) than those of maize plants grown in the same pots (>0.320 atom% “N excess) which would indicate that the non-nodulating beans had tixcd 23-28% of their N (Table 5) but as they formed no nodules this is not likely. Sole maize had similar rJN-cnrichmcnt to the maize intcrcroppcd with non-nodulating bean but cnrichmcnts of maize intcrcroppcd with Nz-fixing bean wcrc much smaller (Table 6) indicating that some of the fixed N had been transfcrrcd from bean to maize. Calculation of N transfer using the equations of Vallis PI al. (1967) and Ta and Faris (1987) indicated that 20 to 35% of the maize N had been dcrivcd from intercropped N,-fixing bean, which amounted to IO-15% of the bean N (Table 7). Enrichments of maize plants intcrcroppcd with N,-fixing bean were reduced, but not significantly less, where they had been inoculated with VA mycorrhiza. The atom% “N excessof maize intercropped with non-nodulating bean was very similar to that of the sole maize, indicating that little N had been transferred. DISCUSSION
Measurement of N-transfer by ‘jN-foliar feeding Transfer of N from bean to intercropped maize was easily detected in both of our foliar feeding exper-
Table 5. Nitrogen fixation in bexr plants grown intercropped with maize on “N-labelled soil calculated by isotope dilution using the maize or the non-nodulatina beans as the reference plant (Extw-iments 3 and 4) ?‘o N tired Treatments
Maize rcfcrcncc
Non-nod reference
Maize reference
58 0. I.3
64 IO I.2
211 0. 9.a
228 II 8.0
84 90 0. 0’ 2.8
89 92 28 23 4.8
a5 100 0. 0’ 9.7
90 103 7 5 9.6
Non-nod reference
Exprrimmt J Maize + bean Maize + non-nod bean SE Experiment 4 Maize + bean Maize + bean + VAM Maize + non-nod bean Maize + non-nod bean + VAM SE ‘Excluded from statistical analysis.
N fixed (mg pot -I’
KENNETHE. GILLERer of.
34.4
Table 6. Growth. mycorrhizal Infection and “N-uptake of (a) bean and (b) maize plants grown as sole or mtercrops oo “N-laklled with or without mycorrhizal inoculation (VAM) (Experiment 4)
N contmt (me pa-‘)
Dry WighI (8 PC-‘) Shoot
Root
Shoot
kan kan + VA41 non-nod bean non-nod bean + VAM
3.3 3.9 1.2 I.? 0.28
1.2 I.1 I .o I.0 0.10
74 86 I5 I3 7.1
Sole maile Sole maize + VAM Mauc + kan Maue + kan + VAM MUX + non-nod kan Maize + non-nod kan + VAM SE
6.4 5.7 4.0 3.5 4.1 2.7 0.34
4.7 5.0 3.2 31 3.3 2.4 0.37
30 27 20 ::
Maize Maize Maize Maize SE
+ + + +
soil
Atom?,'b ‘!S excess % VAM infection
Root
Shoot
Roar
Weighted mean atom% W excess
(a) In beanl 25 26 IO IO 2.8
O+ 18 o* 20 1.6
0.03 I 0.020 0.232 0.255 0.015
0.060 0.W 0.227 0.239 0.019
0.039 0.025 0.243 0.248 0.015
(a) In make* 28 28 24 24 20 15 18 1.4 2.2
5t 4Ot 0) 39 It 38 5.5
0.354 0.381 0.285 0.218 0.374 0.369 0.022
0.‘8 0.;8; O.‘Y 0.G 0.299 0.279 0.013
0.316 0.326 0.252 0.225 0.337 0.320 0.014
*Four maize plants per pot in sole maize treatments, two maize plants per pot in intercrop treatments. Walues excluded from statistical analysis.
in montmorillonite clay where N yields of the nonfixing plants were greater. Whatever the limitations of the methods, it is surprising that although both methods indicate that maize has derived N from bean. no benefit can be seen in increased N uptake by maize. Perhaps similar amounts of N are transferred from maize to bean, but this was not tested in our experiments. Thus N transfer has not contributed to bcttcr growth and N yield of maize despite the severe N limitation for growth imposed in thcsc expcrimcnts and the large amount of N fixed by the beans. Thcrc was no indication that the lcgumc nodulescontributed much directly to N transfer as amounts transfcrrcd wcrc similar with both N,-fixing and non-nodulated beans. and ths proportion of bean N transferred was much less in the case of NJ-fixing beans. i~~c~ll.~urc,r,lt,nt of N-rrctnsfer by ‘5NN-isoropedilution
NJ-fixing bean accumulated roughly three times the amount of N found in maize or non-nodulating bean, indicllting that the amount of N available from the soil was limiting (Tables 4 and 5). The small ‘!N-enrichments of the N,-fixing beans compared to the maize or non-nodulating beans clearly demonstrate that this extra N was from N2-fixation. Despite the large amount of N fixed in the bean plants no benefit in terms of increased N yield was found in the maize plants intercropped with them. In fact in one experiment the only significant difference detected between maize intercropped with the N,-fixing beans or the non-nodulating beans was that the maize had smaller shoot N when intercropped with N,-fixing
beans, presumably due to competition from the larger plants (Table 4). In this experiment the “Nenrichments of maize were virtually identical whether intercropped with N,-fixing or non-nodulating bean (difference ~2%) suggesting that there was no significant transfer of N. We were surprised to find that the “N-enrichment of the non-nodulating beans was much less than that of maize grown in the same pot in both isotope dilution experiments (Tables 4 and 6). The “N-fertilizcr had been added to the soil with the sucrose to stimulate immobilisation of the “N in the microbial biomass, well in advance of the experiment. This should have reduced any possible diferences in the “N-enrichment of N absorbed from the growth medium due to ditl’ercnccs in N uptake pattern of the two crops (Witty, 1983; Giller and Witty, 1987). The seed N content of the two crops was similar (nonnodulating beans 7.6 mg N seed-‘; maize 6. I mg N seed-‘) and cannot have caused the difference in enrichments. The lower “N-enrichment of the nonnodulating beans thus indicates that they were able to obtain N which was less highly enriched in “N, but the source of this N is unclear. We are confident that this was not from N?-fixation as nodules have never been found on roots of this non-nodulating genotype of beans in this or in numerous other glasshouse and field experiments. Perhaps the legume can stimulate mineralisation of N from less highly enriched organic N fractions in the soil, although it is surprising that the “N-enrichments differed so much considering that the roots were closely entangled throughout the
T.thle 7. Nitrogen transfcrrcd from kans to inlercropped maize plants with or withoul inoculation with VA mycorrhiza. calculated using eauations (3) and (4) (Experiment 4) N transferred calculated by using equation (3) (Vallis cf al.. 1967)
Maize Maize Maize Maize SE’
+ + + +
beans beans + VAM non-nod kans non-nod kans + VAM
Amount of N transferred (me pot-‘)
N transferred as % of kan N
N transferred as % of maize N
9 14 I -3 2.5
IO I4 3 -1 3.5
20 31 2
lSlandard error for the firs1 IWO treatments means only.
-1
4.7
N transferred calculated using equation (4) (Ta and Faris. 1987) Amount of N transferred (mg pot-‘) IO I5 -3 I 2.8
X transferred as % of kan N
N transferred as % of maize N
II I5 -13 I 3.5
22 33 -14 2 4.2
345
Nitrogen transfers in b*afi-maize intercrops soil. When non-nodulating bean or maize were used as reference plants for measurement of Nz-fixation in nodulated bean, as expected the estimates differed (Table 5). In this case as the proportion of N estimated to have come from N,-furation was high in both experiments, the difference of iJN-enrichment in the two reference crops had a relatively small effect on the estimates. Differences in the ‘JN-enrichment between reference plants would cause increasing disparity where smaller estimates of N,-fixation are found. Transfer of N from N,-fixing bean to maize can also be calculated by isotope dilution by comparison of the iJN-enrichments of the maize intercropped with N,-fixing bean with either the enrichments of the sole maize or maize intercropped with non-nodulating bean. In this experiment there was little difference in iJN-enrichment of the sole maize and maize intercropped with the non-nodulating bean, but based on the above observation we would favour the use of a cereal intercropped with a non-fixing legume in such experiments to take account of possible effects due to inter-specific competition for N. In this experiment where plant growth was poor due to insect attack, IO-IS% of the bean N, equivalent to 20-30% of the maize N. was estimated to have been transferred (Table 7). As c. 90% of the bean N was estimated to have come from N,-fixation in this experiment, there was little difference in the results obtained using the equations of Vallis cf al. (1967) which takes account of only fixed N transfcrrcd. and that of Ta and Faris (1987) which allows for all sources of the N transfcrrcd from beans (Table 7). Maize intcrcroppcd with N,-fixing bean accumulated slightly, but significantly. more N than maize with non-nodulating bean (Table 7) but no incrcasc in N-transfer due to the presence of VAM was detected by either isotope dilution or total N accumulation. The smaller net N accumulation than the benefit indicated from the isotope dilution calculations of N transfer could have been due to transfer of some N from maize to bean.
Mechanisms of N transfer
The foliar feeding experiments clearly demonstrate underground movement of “N from the beans to the maize within a few weeks. The term “excretion” has been used in the literature to describe such relatively rapid loss of N from the legume roots, but we would suggest, by analogy with carbon losses from roots, the term “rhizodeposition” may be more appropriate (Whipps and Lynch, 1985). It is not possible in such experiments to distinguish between N lost actively or passively in exudates, N losses due to death and senescence of sloughed off cells from roots and nodules and indeed direct transfer of N through mycorrhizal hyphae. In all of these cases the rates of loss and turnover of N in the rhizosphere are likely to be such that they could result in movement of N from the legume to the cereal in days rather than weeks. The use of the term “rhizodeposition” may help to clarify this by not attempting to distinguish active and passive losses of N. Significance of N-transfer in intercrops
Despite the severe N limitation imposed in all of these experiments which was apparent in the
restricted growth and N accumulatioo in all plants of maize and non-nodulating bean, no obvious benefits in total N accumulation of maize were found by intercropping with N,&xing bean. The sensitivity of the iJN-foliar feeding experiments was such that transfer of enriched N was readily detected, but the amounts of N transferred were small and in all cases ~5% of the Nr fixed by beans (Table 3). In the isotope-dilution experiments no transfer of N was detected except where growth of all the maize and bean plants was badly affected by insect infestation, and although up to 35% of the maize N was estimated to have come from N transfer there was no obvious benefit in N yield. Although our initial intention was to identify methods suitable for measurement of N transfer between bean and maize in the field, it seems likely that amounts transferred may be negligible unless a severe limitation is placed on the growth of the beans such as the insect attack seen here. In pot experiments with peas and barley, Wilson and Wyss (1937) only found significant increases in N yield of the cereal when the plant growth was reduced by shading. Many field studies on legumocereal intercrops have found no evidence for N transfer (e.g. Ofori ef al.. 1987; Rerkasem and Rerkasem, 1988) and our results show little benefit from N transfer under conditions of severe N limitation which should increase the demand of the cereal for legume N. Numerous, careful ticld studies will be required to determine how frcqucntly and under what conditions benefits occur due to transfer of N from legumes to intercropped cereals in agriculture. Ac&nowle&rmmr.r-Wc thank J. C. Fear for help with chemical analyses. The Overseas Dcvclopmcnt Administration provided part of the funding for this research.
REFERENCES
Adams M. W.. Coync D. P.. Davis J. H. C., Graham D. H. and Francis C. A. (1985) Common bean (Phuseolus uulguris L.) In Grain Lqume Crops (R. J. Summcrlleld and E. H. Roberts. Eds), pp. 433-476. Collins. London. Conway E. J. (1939) Microdrjiiion Ano1ysi.rand Volumerric Error. Crosby-Lockwood, London. Davis J. H. C.. Giller K. E.. Kipc-Nolt J. A. and Awah F. M. (1988) Non-nodulating mutants in common beans. Crop Science 28. 859-860. Eaglesham A. R. J.. Anyanaba A., Ranga Rao V. and Eskew D. L. (1981) Improving the nitrogen nutrition of maize by intercropping with cowpca. Soil Biology & Biochemislry 13, 169-171. Francis R.. Finlay R. D. and Read D. J. (1986) Vaicular-arbuscular mycorrhiza in natural vegetation systems IV. Transfer of nutrients in intra- and inter-specific combinations of host plants. New fhyrologisr 102, 103-111. Gillcr K. E. and Witty J. F. (1987) Immobilized “N-fertilizer sources improve the accuracy of field estimates of N,-fixation by isotope dilution. Soil Biology CcBiochemisrry 19, 459463. Hewitt E. J. (1966) Sand and Wurcr Culrure Merho& in rhe Study o/ Plunr Nurrbion. Commonwealth Agricultural Bureau, Famham Royal. van Kcsscl C. and Roskoski J. P. (1988) Row spacing effects on N,-fixation. N-yield and soil N uptake of intercropped cowpca and maize. Plum and Soil 111, 17-23.
KES~ZTH E. GIU+R er af.
346
van Kessel C., Singleton P. W. and Hoben H. J. (1985) Enhanced N-transfer from soybean to maize by vesicular arbuscular mycorrhizal (VAM) fungi. Pianr Physiology 79, 562-563.
Ledgard S. F.. Freney J. R. and Simpson J. R. (1985) Assessing nitrogen transfer from legumes to associated grasses. Soil Biology & Biochemisrry 17. 575-577.
Ofori F., Pate J. S. and Stem W. R. (1987) Evaluation of N,-fixation and nitrogen economy of a maize/cowpea intercrop system using “N dilution methods. Pfanr and Soil 102. 149-160.
Papastylianou I. (1988) The “N methodology in estimating N,-fixation by vetch and pea grown in pure stand or in mixtures with oat. Piunr and Soil 107, 183-188. Phillips J. H. and Hayman D. S. (1970) Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection, Transacrions ofrhe Brirish Myrologicul Sociery 55,
I 58-
I6
I.
Rennie R. J.. Rennie D. A. and Fried M. (1978) Concepts of “N usage in dinitrogen fixation studies. In lsoropes in Biological Dinirrogen Fixurion. pp. 107-I 30. International Atomic Energy Agency. Vienna. Rerkasem K. and Rerkasem B. (1988) Yields and nitrogen nutrition of intercropped maize and ricebean (V&IO unih&rn ffhumb.] Ohwi and Ohashi). Planr and Soil IOR. 151-162.
Ta T. C. and Faris M. A. (1987). Effects of alfalfa proportions and clipping frquencies on timothy-alfalfa mixtures. II. Nitrogen fixation and transfer. Agronomy Journal 79, 820-824. Vallis I., Haydock K. P.. Ross P. J. and Henzell E. F. (1967) Isotopic studies on the uptake of nitrogen by pasture plants. III. The uptake of small additions of “N-labelled fertilizer by Rhodes grass and Townsville luame. Ausrralian
JOWMI of Agriculrural
Research 18. 865-877.
Vandermar J. (1989) The Ecology of hercropping. Cambridge University Press, Cambridge. Varley J. A. (1966) Automated methods for the determination of nitrogen, phosphorus and potassium in plant materials. Analysr 91, 119-121. Whipps J. M. and Lynch J. M. (1985) Energy losses by the plant in rhizode position. ANI& Proceedings o/rhe Phyrochemical Sociery of Europe 26, 59-71. Willey R. W. (1979) Intercropping-its importance and research needs. Part I. Competition and yield advantages. Field Crop Absrracrs 32, I-IO. Wilson P. W. and Wyss 0. (1937) Mixed cropping and excretion of nitrogen by leguminous plants. Soil Science Sociery of America Proceedings 11, 289-297. Witty J. F. (1983) Estimating N,-fixation in the field using isN-labelled fertilizer: some problems and solutions. Soil Biology & Biochemisrry 15. 631-639. Witty J. F. and Ritz K. (1984) Slow-release “N fertilizer formulations to measure Nr-fixation by isotope dilution. Soil Bio1og.v & Biochemisrry 16. 657-661.